One of the potential biological warfare agents most feared by civil defense planners today is Bacillus anthracis, or anthrax. This organism makes an effective bioterrorist weapon because it has a high mortality rate, can be readily prepared and stored as spore particles, and delivered over a large area as an aerosol. Thomas V. Inglesby et al., “Anthrax as a biological weapon: Medical and Public Health Management.” JAMA, 281(18) 1735-1745 (1999). This has caused anthrax to be classified as a category A (high priority) agent by the US Centers for Disease Control and Prevention (CDC).
Dissemination of biological warfare agents may occur by aerosol sprays, explosives, or food or water contamination. To be an effective biological weapon, airborne pathogens must be dispersed as fine particles less than 5 μm in size. Advanced delivery systems are not required for the aerosolized delivery of biological agents, which can be delivery by agricultural crop-dusters, aerosol generators on small boats or trucks, backpack sprayers, and even purse-sized perfume atomizers. A biological weapon attack is likely to be covert, and protective measures should be taken when warning is received or once there is suspicion that a biological warfare agent has been or soon will be used. Use of broad spectrum antibiotics is recommended by the CDC for suspected victims of a biological warfare attack, prior to the identification of the specific biological warfare agent used.
The CDC has three categories for biological warfare agents. Category A biological warfare agents are the most serious. The U.S. public health system and primary health-care providers must be most prepared to address these biological agents, which include pathogens that are rarely seen in the United States. High-priority, Category A agents include organisms that pose a risk to national security because they can be easily disseminated or transmitted person-to-person, cause high mortality, with potential for major public health impact, might cause public panic and social disruption, and require special action for public health preparedness. These agents/diseases include: Bacillus anthracis (anthrax), Clostridium botulinum toxin (botulism), Yersinia pestis (plague), Variola major (smallpox), Francisella tularensis (tularemia), and viral hemorrhagic fevers.
Next are Category B biological warfare agents. These are the second highest priority agents, and include those that are moderately easy to disseminate, cause moderate morbidity and low mortality, and require specific enhancements of CDC's diagnostic capacity and enhanced disease surveillance. These agents/diseases include: Coxiella burnetti (Q fever), Brucella species (brucellosis), Burkholderia mallei (glanders), ricin toxin from Ricinus communis (castor beans), the epsilon toxin of Clostridium perfringens, and Staphylococcus enterotoxin B.
Finally, there are Category C biological warfare agents. These are the third highest priority agents, and include emerging pathogens that could be engineered for mass dissemination in the future because of availability, ease of production and dissemination, and potential for high morbidity, mortality, and major health impact. These agents/diseases include: Nipah virus, the hantaviruses, the tickborne hemorrhagic fever viruses, the tickborne encephalitis viruses, yellow fever, and Mycobacterium tuberculosis (tuberculosis).
An example of a Category A biological warfare agent is B. anthracis. B. anthracis is an aerobic gram-positive rod that commonly infects herbivores causing a serious and often fatal disease. Spores are produced at temperatures below 30° C. in soil and on inanimate objects, but not in living tissues. Spores can resist to temperatures above 100° C. for limited periods of time, making them highly persistent. Humans can acquire the disease by contact with infected animals or infected animal products. Once inside the body, spores germinate into “vegetative” or actively dividing cells. Direct contact between spores or infected tissues and broken skin results in cutaneous anthrax. Within two to five days of exposure, a small papule develops, followed by a necrotic ulcer surrounded by oedema. Death following treatment is very rare but untreated persons have a mortality rate near 20%. Ingestion of undercooked meat may result in gastrointestinal anthrax. Nausea, vomiting, and gastrointestinal bleeding ensue within 12 to 18 hours, leading to haemorrhagic lymphadenitis. Spread to the bloodstream and subsequent death can occur.
Inhalation anthrax is the most likely form of the disease from biological warfare use, and is the most dangerous. Much of the currently available data on human reaction to inhalation anthrax is a result of the accidental aerosolized release of anthrax spores from a military microbiology facility in Sverdlovsk in the former Soviet Union in 1979, which resulting in at least 79 cases of anthrax infection and 68 deaths. Inhalation of anthrax spores causes the most serious form of infection, resulting in influenza-like illness within one to five days of exposure. Early diagnosis of inhalational anthrax is difficult and requires a high index of suspicion, as the first stage of the disease is relatively benign. The second stage develops abruptly, however, with sudden fever, dyspnea, diaphoresis, and shock, leading to massive lymphadenopathy and hemorrhagic meningitis. In the second stage of illness, cyanosis and hypotension progress rapidly; death sometimes occurs within hours. Franz D. R., et al., “Clinical recognition and management of patients exposed to biological warfare agents.” JAMA, 278, 399-411 (1997). Inhaled anthrax is nearly always fatal because of the rapid progression of the disease and the benign appearance of the initial symptoms. Fritz et al., Lab. Invest., 73, 691-702 (1995).
The pathogenesis of infection by B. anthracis is not yet completely understood. At its most basic level, extensive replication in the blood is generally what kills patients who succumb to anthrax. B. anthracis's ability to expand so successfully derives partially from its production of virulence factors that can profoundly depress the immune system. The major virulence factors of B. anthracis are a poly-D-glutamic acid capsule, that inhibits ingestion and destruction by the immune system's macrophages and neutrophils, and exotoxin. Exotoxin is composed of three protein components; protective antigen (PA), lethal factor (LF), and edema factor (EF). Ballard et al., PNAS, 93, 12531-12534 (1996). These proteins cooperate but are not always joined together. Protective antigen binds to cell surfaces to trigger the formation of an endosome which is used to transport edema factor and lethal factor across the endosomal membrane into the cytosol of the target cell. Edema factor upsets the controls on ion and water flow across the cell membrane, promoting swelling. Lethal factor is a protease whose precise mechanism of action remains unknown. Brossier et al., Infect. Immun., 68, 1781-1786 (2000). However, it is known that lethal toxin inhibits macrophages from releasing the immune messengers interleukin-1 (IL-1), interleukin-2 (IL-2), gamma interferon, and tumor necrosis factor alpha (TNF-α).
The macrophage plays a key role in the success or failure of many pathogenic organisms utilized as biological warfare agents, such as B. anthracis. Macrophages are the first cells to interact with B. anthracis via phagocytosis. Vesicles derived from the phagosomal compartment of alveolar macrophage are the primary sites of spore germination in a murine inhalation model. Guidi-Rontani et al., Mol Microbiol., 31, 9-17 (1999). Macrophages induce host defense responses such as cytokine secretion and cell mediated cytotoxicity against infection by pathogens such as anthrax. The early onset of toxin gene expression after germination is a key determinant in the macrophage response. The LF protein is lytic for macrophages and sublytic doses of LF cause a reduction of nitric oxide (NO) and tumour necrosis factor (TNF) production. Pellizzari et al., FEBS Letters, 462, 199-204 (1999). On the other hand, Hanna et al showed that sublytic concentration of LF could increase the production of IL-1 by activated macrophages. Hanna, et al., PNAS, 90, 10198-10201 (1993). Along with macrophage activation, Natural Killer (NK) and T cells also take an active part in protection against diseases such as anthrax. After first contact with antigen, a rise in the NK cell activity is observed, followed by its pronounced suppression. Soloklin et al., Zhurnal Mikrobiologii Epi. Immuno., 5, 72-76 (1995). The use of protective antibody against pathogens such as anthrax increases the duration of NK cell activity. This suggests a major role of macrophages and NK cells in the host defense mechanisms against biological warfare agents such as anthrax.
On a separate subject, β-glucan is a complex carbohydrate which has shown potential as an immunomodulator. It is generally derived from several sources, including yeast, bacteria, fungi and cereal grains. These sources provide β-glucans in a variety of mixtures and purities, and with a variety of different chemical structures. The structural diversity of β-glucans results from the fact that glucose molecules can be linked together in many different ways, resulting in compounds with different physical properties. For example, β(1,3)-glucans derived from bacterial and algae are linear, making them useful as a food thickener. Lentinan (from Lentinus edodes, Basidiomycete family) is a high MW β-glucan with single glucose branches linked (1,6)-β off of the (1,3) backbone every three residues. Schizophyllan (from Schizophyllum commune, Basidiomyctes family) is a similar β-glucan. Beta-glucans from barley, oat, or wheat have mixed (1,3)- and (1,4)-D-linkage in the backbone, but no (1,6)-β branches, and are generally of high molecular weight. The frequency of side chains, known as the degree of substitution or branching frequency, regulates secondary structure and solubility. Beta-glucans derived from yeasts have a backbone chain of β(1-3) linked glucose units with a low degree of inter and intra-molecular branching through β(1-6) linkages. Based on extensive published research it is widely accepted that baker's yeast (Saccharomyces cerevisiae) is a preferred source of β(1,3)-glucan, based on the purity and activity of the product obtained.
The cell wall of S. cerevisiae is mainly composed of β-glucans, which are responsible for its shape and mechanical strength. While best known for its use as a food grade organism, yeast is also used as a source of zymosan, a crude insoluble extract used to stimulate a non-specific immune response. Yeast zymosan serves as a rich source of β(1,3)-glucan. Yeast-derived β(1,3)-glucan appears to stimulate the immune system, in part by activating the innate immune system as part of the body's basic defense against fungal infections. Yeast β(1,3)-glucan is a polysaccharide composed primarily of β(1-3)-linked glucose molecules with periodic β(1-3) branches linked via β(1-6) linkages and is more formally known as poly-(1,6)-β-D-glucopyranosyl-(1,3)-β-D-glucopyranose.
Various forms of β(1,3)-glucan have been prepared, with differing properties. These different forms vary in terms of their purity, particle size, and solubility. One of the larger and less soluble forms of β-glucan are whole glucan particles. Whole glucan particles are the remnants of the yeast cell wall prepared by separating growing yeast from its growth medium and subjecting the intact cell walls of the yeast to alkali, and removing unwanted proteins and nucleic acid material, as well as the mannan component of the cell wall. Normally, what remains is purified, 2-5 micron, spherical β-glucan particle. Whole glucan particles may be obtained from any glucan-containing fungal cell wall source, but the preferred source is S. cerevisiae. Methods of producing whole glucan particles are known in the art and are disclosed in U.S. Pat. Nos. 4,810,646, 4,4992,540, 5,037,972, 5,082,936, 5,250,436, and 5,506,124, the contents of which are incorporated herein by reference.
Another, more processed form of β(1,3)-glucan is PGG glucan, which is an abbreviation of the full chemical name, poly-(1,6)-β-D-glucopyranosyl (1,3)-β-D-glucopyranose. PGG glucan (PGG), in a particular formulation in saline, goes by the trademark Betafectin®. Generally, neutral underivatized β(1,3)-glucans are not soluble in physiological media due to their tendency to form tightly associated triple helix fibrils which resist hydration. PGG is prepared from whole glucan particles through a series of acid, alkaline and neutral treatment steps to yield a conformationally pure, triple helical soluble glucan preparation which can be maintained in a clear solution at neutral pH. Methods of producing PGG are known in the art and are disclosed in U.S. Pat. Nos. 5,322,841, 5,811,542, 5,663,324, 5,633,369, and 5,817,643, the contents of which are incorporated herein by reference. The soluble glucans produced by this process are branched polymers of glucose, containing β(1-3) and β(1-6) linkages in varying ratios depending on the source organism and the exact processing conditions used. The average weight of PGG-glucan molecules is generally about 5,000 to 500,000 daltons.
Microparticulate glucan is glucan that is refined to produce smaller fragments of whole glucan particle. Methods of producing microparticulate β-glucan are disclosed in U.S. Pat. Nos. 5,223,491, 5,397,773, 5,576,015, 5,702,719, and 5,705,184, the contents of which are incorporated herein by reference. The beta (1,3) glucan used to prepare microparticulate glucan is isolated from yeast cell walls by conventional methods known by those of ordinary skill in the art. Microparticulate glucan generally has average particle size is preferably about 1.0 microns or less, and more preferably about 0.20 microns or less.
Beta glucans possess a diverse range of activities. The ability of β-glucan to increase nonspecific immunity and resistance to infection is similar to that of endotoxin. Early studies on the effects of β(1,3)-glucan on the immune system focused on mice. Subsequent studies demonstrated that β(1,3)-glucan has strong immunostimulating activity in a wide variety of other species, including earthworms, shrimp, fish, chicken, rats, rabbits, guinea pigs, sheep, pigs, cattle, and humans. For a review, see Vetvicka V. “β-glucans as immunomodulators”, JANA, 3, 24-31 (2001). Based on these studies it has been concluded that β(1,3)-glucan represents a type of immunostimulant that is active across the evolutionary spectrum, likely representing an evolutionarily-conserved innate immune response directed against fungal pathogens. However, despite extensive investigation, no consensus has been achieved on the source, size, and form of β(1,3)-glucan ideal for use as an immunostimulant.
There have been several studies on the use of β-glucans to prevent infection, primarily in the context of surgical sepsis. For example, Williams et al. assessed the role of combined immunomodulation with β-glucan and antibiotic (gentamycin) in the treatment of experimental sepsis. Williams et al., “Synergistic effect of nonspecific immunostimulation and antibiotics in experimental peritonitis” Surgery, 102(2), 208-14 (1987). This particular study noted that β-glucan treatment alone after E. coli inoculation was expected to have no beneficial effect on long-term survival. A similar study was conducted by Kaiser, who used PGG glucan and cefazoline antibiotic synergistically to prevent staphylococcal wound infection. Kaiser A. B, Kemodle D. S., “Synergism between poly-(1-6)-beta-D-glucopyranosyl-(1-3)-beta-D-glucopyranose glucan and cefazolin in prophylaxis of staphylococcal wound infection in a guinea pig model”, Antimicrob. Agents Chemother., 42(9), 2449-51 (1998).
The molecular mechanism of action of β-glucan appears to involve specific β-glucan receptor binding sites on the cell membranes of immune cells such as neutrophils and macrophages. Mannans, galactans, α(1-4)-linked glucose polymers and β(1-4)-linked glucose polymers have no avidity for this receptor. Recent data suggests that CR3, the receptor for C3 complement protein, serves as a major receptor for β-glucans. Ligand binding to the β-glucan receptor results in complement activation, phagocytosis, lysosomal enzyme release, and prostaglandin, thromboxane and leukotriene generation providing a more functionalized innate immune system to protect against a wide array of pathogenic challenges.
The recent increased threat of bioterrorism, which could result in the widespread dissemination of one or more pathogenic organisms, has increased our awareness that we have relatively few prevention and treatment options available for protecting the U.S. public. In 1970, a World Health Organization (WHO) expert committee estimated that the release of 50 kg of anthrax from an aircraft over a developed urban population of 5 million would result in 250,000 casualties, 100,000 of whom could be expected to die without treatment. Health Aspects of Chemical and Biological Weapons, Geneva, Switzerland, WHO; 98-99 (1970). A 1993 report by the US Congressional Office of Technology Assessment estimated that between 130,000 and 3,000,000 deaths would follow from the aerosolized release of 100 kg of anthrax spores upwind of the Washington, D.C. area—lethality matching or exceeding that resulting from the detonation of a hydrogen bomb. Office of Technology Assessment, US Congress, Washington, D.C., “Proliferation of Weapons of Mass Destruction”, US Government Printing Office, 53-55 (1993).
The first evidence of a terrorist release of anthrax as a biological weapon would likely be patients seeking medical treatment for symptoms of inhalational anthrax. The sudden appearance of a large number of patients in a city or region with an acute-onset flu-like illness and fatality rates of 80% or more, with nearly half of all deaths occurring within 24-48 hours, would indicate the highly likelihood of an anthrax or pneumonic plague release. Rapid diagnostic tests for diagnosing anthrax, such as enzyme-linked immunosorbent assay for protective antigen and polymerase chain reaction, are available only at national reference laboratories. Many other biological warfare agents would be equally difficult to respond to in a timely fashion.
Conventional anti-microbial therapies, such as antibiotics, can be useful to treat some bioterroristic pathogens, but are generally not useful for protecting the public from infection until after exposure. Antibiotics such as ciprofloxacin (a fluoroquinolone antibiotic) and doxycycline (a tetracycline antibiotic) are useful for treating anthrax; however, even the use of multiple antibiotics is often not enough to prevent symptomatic patients from succumbing to infection. Furthermore, reports have been published of a B. anthracis strain that has been engineering by Russian scientists to resist antibiotics. Prophylactic administration of vaccines, such as that disclosed by Ivins et al. in U.S. Pat. No. 6,387,665, provides another means to protect the public from infection. For example, a US anthrax vaccine, made up of an inactivated cell-free product, has been mandated for all US military active- and reserve-duty personnel. Unfortunately, a single vaccine is only able to protect against infection by a single microorganism and does not provide broad protection against multiple possible pathogenic terrorist threats. Further, widespread vaccination is not recommended for protecting the general public as there is limited availability of vaccine, and debate as to whether the risk of adverse side-effects justifies its general use. The timeframe for the development of safe and effective treatment and providing cost-effective delivery of these treatments to a large military or civilian population are also significant issues. Thus, what is clearly needed is a method of protecting against biological warfare which increases survival when administered both before and after exposure, and which provides effective defense against a wide variety of possible biological warfare agents, as well as being inexpensive to provide to the general public, and readily capable of being stored for extended periods.